Mass spectrometry is now a well-established technique for analyzing substances by separating ions due to their differing mass to charge ratios. A wide variety of mass spectrometers and ionization techniques are known. The present invention is particularly, although not exclusively, concerned with electrospray-generated ions, and more particularly the use of this ionization technique with large organic molecules.
Mass spectrometry of electrospray-generated ions is a very sensitive technique for identification and quantification of trace compounds at low concentrations. In particular, it is now known that electrospray ionization techniques generate multiply charged ions allowing analysis with mass spectrometers with limited mass ranges. Many organic compounds can be ionized so to have multiple charges. For example, multiply charged ions of peptides formed from protein digestion by the enzyme trypsin have been shown to be useful for sequence determination following product ion MS/MS scans, as is described by Covey et. al. in U.S. Pat. No. 5,952,653. A product ion scan is now a well known analysis technique in mass spectrometry, in which a precursor ion is selected, caused to fragment (usually by acceleration into a collision cell), and then the fragments are scanned to determine the fragments or products generated from the selected precursor, which can give information about the structure of the precursor. One difficulty however is that it can be a challenge to identify low concentration multiply charged peptides in the single MS survey scan due to the presence of singly charged chemical noise that is often present in such scans. MS/MS techniques such as precursor ion and neutral loss scanning can partly offset the chemical noise problem by introducing an additional degree of specificity to the survey scans (a precursor ion scan holds the selected product or fragment ion mass to charge ratio fixed and scans to identify precursor ions that generate such the selected product of fragment ion; a neutral ion scan maintains a fixed mass difference between a selected precursor ion and a selected product/fragment ion). The utility of these scans however requires some prior knowledge of the sample, which is not always the case. For example, to carry out a meaningful precursor scan, it is necessary to have some knowledge of fragment ions that might be generated. Thus, analysis of analytes that produce multiply charged fragment ions can generate some unique problems.
Linear ion traps have been reported to discriminate against higher m/z ions under conditions in which the overall charge density is high. This is due to the fact that, at a given RF voltage or trapping q-value, the potential wells for higher m/z ions are shallower than those for ions with lower m/z values [Tolmachev et. al. Rapid Commun. Mass Spectrom. 14, 1907–1913(2000)]. This is true for both linear ion traps with two-dimensional radio frequency trapping fields and conventional ion traps with three-dimensional trapping fields. However, this does not address the problem of differentiating between multiply charged ions (often desired analyte ions) and singly charged ions (often unwanted chemical noise) with the same m/z. The inventor of the present invention has found that the population of multiply charged ions of a given m/z can be enhanced relative to the population of singly charged ions at the same m/z. This then makes it possible to identify low concentration multiply charged ions in what would normally be much more concentrated singly charged chemical noise.